The present invention relates to a population of expanded and re-differentiated adult islet beta cells capable of both storing insulin in physiological amounts and secreting insulin in response to glucose.
Type I diabetes is caused by the autoimmune destruction of the pancreatic islet insulin-producing beta cells. Insulin administration does not prevent the long-term complications of the disease, since the optimal insulin dosage is difficult to adjust. Replacement of the damaged cells with regulated insulin-producing cells is considered the ultimate cure for type 1 diabetes. Pancreas transplantation has been successful but is severely limited by the shortage of donors. With the development of new islet isolation and immunosuppression procedures, significant success has been reported using islets from 2-3 donors per recipient (Shapiro A M, Lakey J R, Ryan E A et al. New Engl J Med 2000; 343:230-238). This progress underscores the urgent need for developing alternatives to human pancreas donors, namely abundant sources of cultured human β cells for transplantation.
Terminally differentiated, postmitotic islet cells are difficult to expand in tissue culture. Adult and fetal human islet cells grown on HTB-9 matrix in RPMI 1640 medium containing 11 mM glucose, and supplemented with 10% FBS and hepatocyte growth factor, were shown to proliferate at the most for 10-15 population doublings, after which they underwent senescence. The replication span could not be extended by expression of the catalytic subunit of human telomerase (hTERT), which was introduced into the cells with a retrovirus (Halvorsen T L, Beattie G M, Lopez A D, Hayek A, Levine F. J Endocrinol 2000; 166:103-109). Due to massive cell death, this method resulted in a 3-4 expansion of the islet cell mass.
An alternative to forced expansion of post-mitotic β cells is the induction of differentiation of stem/progenitor cells, which have a natural self-expansion capacity, into insulin-producing cells. The directed differentiation of embryonic stem cells has generated cells that only produce low amounts of insulin, compared to β cells, and their potential use in transplantation has met with ethical objections, as well as concerns regarding risk of teratomas.
Adult stem cells have also been differentiated into insulin-producing cells. However, the efficiency of expansion of these cell types in tissue culture and their rate of differentiation into insulin-producing cells need to be greatly improved to allow generation of significant cell numbers for transplantation.
It has been clearly demonstrated that committed cells can be at least partly reprogrammed with dominant genes that activate a cascade of developmental events. U.S. Pat. Appl. No. 20050244966 to the present inventors teaches the reprogramming of fetal hepatic cells into beta-like insulin-producing cells by expression of dominant transcription factors, such as Pdx1, that direct the development of endocrine pancreas. The human fetal liver cells were induced to produce and store mature insulin in significant amounts, about a third of those produced by normal β cells, release it in response to physiological glucose levels, and replace β-cell function in STZ-diabetic nonobese-diabetic severe combined immunodeficient (NOD-scid) mice. The modified cells expressed multiple β-cell genes.
Islet cells have been expanded ex vivo in the presence of epidermal growth factor and nerve growth factor. Although these cells show high insulin content, they do not secrete insulin in response to glucose (Lechner A. et al., Biochem Biophys Res Commun 327:581-588, 2005).
There is thus a widely recognized need for, and it would be highly advantageous to have, abundant sources of cultured human β cells capable of producing physiological concentrations of insulin for transplantation devoid of the above limitations.
A number of factors have been shown to promote both β-cell proliferation and differentiation in tissue culture. Members of the growth hormone family, including placental lactogen (PL), growth hormone (GH) and prolactin (PRL), induce replication in neonatal rat islet cells. Significant mitogenic effects of hepatocyte growth factor (HGF) have been observed on human fetal and adult islets and mouse islets. In the presence of activin A or nicotinamide, HGF has been shown to stimulate β-cell differentiation in cultured fetal pancreatic islets as well as a pancreatic cell line. Glucagon-like peptide 1 (GLP-1), and its more stable analog exendin-4, have been shown to stimulate β-cell proliferation and to induce insulin gene expression in a pancreatic cell line. Members of the epidermal growth factor (EGF) family, including EGF, TGFα and betacellulin, have also been shown to stimulate β-cell proliferation and differentiation. Betacellulin is a potent mitogen for a number of cell types, including β cells. It was shown to increase islet neogenesis in alloxan and STZ-treated mice, and accelerate islet-regeneration in 90%-pancreatectomized rats [Li L, et al., Endocrinology 2001; 142:5379-5385].
In a number of rodent models, betacellulin, in combination with other factors was shown to comprise differentiation capabilities. Thus, in rodent pancreatic cell lines, betacellulin was shown both alone and in combination with other factors to induce differentiation of insulin producing cells. In addition, Pdx1 expression, combined with betacellulin treatment induced insulin expression in the mouse glucagonoma cell line alpha TC1-6, and the rat intestinal cell line IEC-6. Expression of neuroD, combined with in vivo treatment with betacellulin, induced conversion of mouse liver cells into insulin-producing cells.
Betacellulin has been shown to induce proliferation of a number of human pancreatic cell types. For example, betacellulin was shown to stimulate proliferation of adult human pancreatic duct cells [Rescan et al., Laboratory Investigation (2005) 85, 65-74].
In combination with other factors, betacellulin may also enhance differentiation into insulin-producing cells. For example, together with activin A, betacellulin induced differentiation of human fetal pancreatic cells into insulin producing cells. However, while activin A was shown to comprise the differentiating activity, betacellulin was shown to comprise a mitogenic activity only [Demeterco et al., Journal of Clinical Endocrinology and metabolism, 2000, Vol. 85, No. 10 3892-3897].
U.S. Pat. Appl. No. 20040132679 teaches the administration of betacellulin in conjunction with islet cell differentiation transcription factors for the treatment of type I Diabetes. These factors were shown to enhance the differentiation of a population of hepatic cells into insulin producing cells. However, when administered alone, betacellulin had no effect on the serum glucose of STZ induced diabetic mice, indicating that betacellulin alone was not able to differentiate the hepatic cells, but rather had a mitogenic activity thereon. Furthermore, therapeutic efficacy was shown only for the in-vivo administration of the islet cell differentiation promoting factors.
In conclusion, it has never been demonstrated that betacellulin per se has insulin producing capabilities in human pancreatic cells, only mitogenic capabilities.
It has been suggested that Neurogenin 3 (Ngn3) is a key proendocrine transcription factor. This has been demonstrated by the absence of endocrine cells in mice lacking Ngn3, by lineage tracing analyses, and by ectopic expression of Ngn3. Ngn3 is involved in the lateral specification events, which control the choice between the endocrine and exocrine cell fates by mutual signaling between neighboring cells through the Notch pathway. Terminal differentiation of β cells requires shut off of Ngn3 expression.
Identification of an insulin cell differentiating factor for an expanded culture of human beta cells would be highly advantageous for cell transplantation in the treatment of diabetes.